Many molecules, both non-organic and organic, show fluorescence emission, especially with excitation using high energy radiation. Similarly, the fluorescence emitted is similar to that given off by normal single-photon excitation. In most cases the electron ends up in the same excited state as with normal single-photon excitation before it drops down to the ground state. The electron is then brought into its excited state with two photons of half the required energy, arriving simultaneously. With transmitted light illumination, a small Stokes shift may make it impossible to illuminate a fluorochrome at its excitation peak and to observe the fluorescence colour at its emission peak.Ĭonfocal laser scanning fluorescence microscopy enables two-photon excitation with photons of longer wave-length than the emitted light.

Fluorochromes with a large Stokes shift are easy to excite and to observe their emission in a fluorescence microscope. The difference in wavelengths between the excitation and emission peaks is referred to as the Stokes shift. This short duration (nanoseconds) distinguishes it from other forms of luminescence such as phosphorescence. The process of absorption of photons is extremely rapid and is immediately followed by a return to lower energy states, which is then accompanied by emission of photons, which can be observed if light is emitted in the visual range. When a fluorochrome absorbs light, energy in the form of photons is taken up, leading to excitation of electrons to higher energy states. A synoptical table of fluorescence microscopy techniques gives relevant information about microscopes, objectives, fluorochromes, light sources and filter systems concerning the fields of application and the methods referring to this.įluorescence is a molecular phenomenon in which a substance absorbs light (excitation) and radiates light of longer wavelength (emission) for a very short period of time. This course of development is described chronologically in the present contribution. Leica Wetzlar in developing the optical instruments required. The progress in incident light fluorescence microscopy was particularly determined by the research work of Ploem and his function as trend setter and also by the forward strategy of Leitz resp. This was the hour of birth of incident light fluorescence microscopy.ĭuring its course of nearly 40 years of application and development this technique became one of the basic tools for routine and research work in biology, medicine, science and industry. But with its increasing importance to histology, cytology, molecular biology and immuno-diagnosis the demand for a fundamental improvement of illumination and observation techniques came up more and more. This was due to its limited rangers of application in those days.

collector lenses, illuminators, beamsplitters, objectives, tube lenses and eyepieces.Ĭompared with the situation of today, fluorescence microscopy was for the first time applied with the use of transmitted light and darkfield microscopy. To perform fluorescence in a microscope the following requirements have to be met: powerful energy sources (highpressure mercury arc lamp, halogen lamp, etc.), adequate transmission filter systems (filterblocks) selecting the excitation light and the emitted radiation perfectly, and, last but not least, optical parts and outfits suitable for fluorescence, i.e. This phenomenon is the basic element of fluorescence microscopy and its application.īesides the 'classical' excitation of fluorescence in a light microscope it is possible today to obtain the same emission effect via the modern technology of confocal laser scanning microscopy by an excitation with two or multiple photons having longer wavengths than those of the emitted ones.įluorescence occurs either as autofluorescenc of biological and/or inorganic structures or as so called secondary fluorescence after a treatment of the specimen with special dyes (fluorochromes, fluorescent markers). Fluorescence is a process where a substance after having absorbed light (photons) emitts a radiation the wavelength (colour) of which is longer than that of the absorbed light, and where this emission stops immediately after cessation of the excitation.

Half life blue shift lp